The objective of drug targeting research is to improve the effectiveness of therapeutic drugs by delivering them directly to the targeted tumor sites and allowing a more effective dosing at these sites, thereby reducing non-tumor-related side effects. Another objective is to achieve an absolute accretion of the therapeutic agent at the target site thereby increasing the target/non-target ratio.
Different targeting vectors comprising diagnostic or therapeutic agents conjugated to a targeting moiety for selective localization have long been known. Examples of targeting vectors include diagnostic agent or therapeutic agent conjugates of targeting moieties such as antibodies or antibody fragments, cell- or tissue-specific peptides, hormones and other receptor binding molecules. For examples, antibodies against different determinants associated with pathological and normal cells, as well as associated with pathogenic microorganisms, have been used for the detection and treatment of a wide variety of pathological conditions or lesions. In these methods, the targeting antibody is directly conjugated to an appropriate detecting or therapeutic agent as described, for example in, Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and 4,818,709, the disclosures of all of which are incorporated herein by reference.
One of the problems encountered in direct targeting methods is that a relatively small fraction of the conjugate actually binds to the target site, while the majority of the conjugate remains in circulation and compromises in one way or another the function of the targeted conjugate. Other problems include high background and low resolution when a diagnostic agent is administered and marrow toxicity or systemic side effects when a therapeutic agent is attached to a long circulating targeting moiety.
Pretargeting methods have been developed to increase the target:background ratios of the detection or therapeutic agents. Examples of pretargeting and biotin/avidin approaches are described, for example, in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226 (1988); Hnatowich et al., J. Nucl. Med. 28:1294 (1987); Oehr et al., J Nucl. Med. 29:728 (1988); Klibanov et al., J. Nucl. Med. 29:1951 (1988); Sinitsyn et al., J. Nucl. Med. 30:66 (1989); Kalofonos et al., J. Nucl. Med. 31:1791 (1990); Schechter et al., Int. J. Cancer. 48:167 (1991); Paganelli et al., Cancer Res. 51:5960 (1991); Paganelli et al., Nucl. Med. Commun. 12:211 (1991); Stickney et al., Cancer Res. 51:6650 (1991); and Yuan et al., Cancer Res. 51:3119 (1991); all of which are incorporated by reference herein in their entireties.
In pretargeting methods, a primary targeting species (which is not bound to a diagnostic agent or therapeutic agent) comprising a first targeting moiety which binds to the targeting site and a binding site that is available for binding by a subsequently administered second targeting species is targeted to an in vivo target site. Once sufficient accretion of the primary targeting species is accomplished, a second targeting species comprising a diagnostic or therapeutic agent and a second targeting moiety, which recognizes the available binding site of the primary targeting species, is administered.
An illustrative example of pretargeting methodology is the use of a biotin-(strept)avidin system to administer a cytotoxic radioantibody to a tumor. In the first step, a monoclonal antibody targeted against a tumor-associated antigen is conjugated to avidin (or biotin) and administered to a patient who has a tumor recognized by the antibody. In the second step, the therapeutic agent, via its attached biotin (or avidin), is taken up by the antibody-avidin (or -biotin) conjugate pretargeted to the tumor.
However, difficulties have arisen in the applications of biotin-avidin or (strept)avidin system during pretargeting. First of all, unless properly constructed, radiolabeled biotins may be subject to plasma biotinidase degradation. Furthermore, when conjugated to antibodies, strept/avidin and avidin can generate anti-strept/avidin antibodies in a patient. Finally, the potential effects of endogenous biotin during in vivo pretargeting can lead to the disappearance of biotin binding expression because of saturation by biotin. This happened, for example, when one strept/avidin-conjugated antibody localized in a nude mouse xenograft became saturated with biotin. Rusckowski et al., Cancer 80:2699-705 (1997). A three-step strategy involving administration of biotinylated monoclonal antibody, avidin, followed by radiolabeled biotin alleviates some of the drawbacks; however, this procedure is considered complex for imaging and does not address immunogenecity.
Another recognized example of pretargeting method involves the use of the bispecific antibody-hapten recognition system which uses a radiolabeled hapten and a bispecific antibody in place of (strept)avidin and biotin. Barbet, J. et al. Cancer Biother. Radiopharm. 14:153-166 (1999); Karacay, H. et al., Bioconj. Chem. 11: 842-854 (2000); Gautherot, E. et al., J. Nucl. Med. 41:480-487 (2000); Lubic, S. P. et al., J. Nucl. Med. 42:670-678 (2001); Gestin, J. F. et al., J. Nucl. Med. 42:146-153 (2001). The hapten is often a coordination complex, for example, indium-DTPA. The bispecific antibody is the product of linking two antibodies or antibody fragments against separate determinants, the hapten and a tumor marker such as carcinoembryonic antigen. In addition to the need to prepare bispecific antibodies, this approach may suffer from lower affinities. The affinity of an antibody for its hapten, particularly for a monovalent one, is orders of magnitude lower than that of (strept)avidin for biotin. Mathematical modeling has shown that a high affinity between an antibody and its hapten is an important determinant of successful pretargeting. Zhu, H. et al., J. Nucl. Med. 39:65-76 (1998).
As an alternative to the biotin-avidin and bispecific antibody-hapten systems for pretargeting, single-stranded oligomers, such as peptide nucleic acid (PNA), have been used. Single-stranded oligomers bind specifically to their complementary single-stranded oligomers by in vivo hybridization. A single-stranded PNA bound to a targeting moiety is first administered to a patient, followed by the single-stranded complementary PNA radiolabeled with a diagnostic agent. An example of this methodology is described in Rusckowski et al., Cancer 80:2699-705 (1997). An optional intermediate step can be added to the two-step method by administration of a clearing agent. The purpose of the clearing agent is to remove circulating primary conjugate which is not bound at the target site. This is disclosed by Griffiths et al., in U.S. Pat. No. 5,958,408, which is incorporated herein by reference.
Chemical modifications to the backbone of these single-stranded oligomers for attachment to radionucleotides are usually required to improve nuclease stability and decrease protein binding affinities. The influence of three distinct chemical modifications to one 18 mer phosphorothioate DNA to permit labeling with 99mTc have been compared in vitro and in vivo in mice. Zhang, Y. M. et al., Eur. J. Nucl. Med. 27:1700-1707 (2000). While the association rate constant for hybridization was found to be independent of labeling method, both cellular accumulations in culture and the pharmacokinetic behavior of the radiolabel in normal mice was strongly influenced by the labeling method.
These in vivo properties of oligomers may possibly be influenced by changes in their chain length and/or base sequences. Conceivably, the pharmacokinetics of an oligomer may thereby be modified in a useful manner if the influences of chain length and base sequence were to be understood. Despite this possibility (and as in the case of the chemical modifications), these additional influences have almost entirely gone uninvestigated thus far. In part, this may be attributed to constraints placed on these parameters by the application. For example, antisense chemotherapy is thought to achieve efficacy usually by the hybridization of a short, single-chain oligomer with a base sequence complementary to that of its mRNA target. Hnatowich, D. J., J. Nucl. Med. 40:693-703 (1999). The base sequence, and to an extent the chain length as well, are thus restricted to those providing the desired hybridization. Nevertheless, there are combinations of bases that have received attention. One example is the presence of a G-quartet (i.e. four guanine bases in a row) in either phosphodiester or phosphorothioate DNAs. Shafter, R. H. et al., Biopoly (Nucleic Acid Sci.) 56:209-227 (2001). In the case of these chemical forms of DNAs at least, the stacking of the guanine bases provides the oligonucleotides with a particular three dimensional quadruplex structure. This structure is apparently responsible for a variety of sequence-specific effects with significance to various biological processes. Shafter, R. H. et al., Biopoly. (Nucleic Acid Sci) 56:209-227 (2001). Another example is the CpG motif, a cytosine base followed immediately by a guanine, that has been shown to be immunostimulatory. Zhao, Q. et al., Antisense Nucleic Acid Drug Dev. 7:495-502 (1997). The influences of these sequences, if any, on pharmacokinetics has yet to be established.
A variety of other published reports have appeared concerning the in vitro influences of oligomer chain length and sequence. Cytotoxicity in one cell line of phosphodiester DNAs composed entirely of guanine and thymidine bases was found to require at least a chain length of 20 bases and the cytotoxicity disappeared with the introduction of adenines or cytosines at either end. Morassutti, C. et al., Nucleosides & Nucleotides 18:1711-1716 (1999). The efficiency with which PNAs initiated transcription and gene expression in cells was found to be optimum at chain lengths of 16 to 18 bases. Wang, G. et al., J. Mol. Biol. 313:933-940 (2001). Rat liver homogenates have been used ex vivo to investigate the metabolism of a series of phosphorothioate DNAs differing in chain length and base sequence. Crooke, R. M. et al., J. Pharm. Exp. Therapeutics 292:140-149 (2000). All oligomers were degraded primarily by 3 ′ exonucleases with the rate of metabolism increasing with increasing chain length. The rate and extent of nuclease metabolism was also related to base sequence in that pyrimidine-rich oligonucleotides were more labile. This particular investigation was unusual in that the influence of sterioisomerism was also studied. The metabolism rate was found to be more rapid for one of the diasterioisomers than the other with mixtures being digested at rates in between. Finally, a recent report described the influence of base sequence on reactivity of the phosphodiester bond in RNAs. Kaukinen, U. et al., Nucl. Acids Res. 30:468-474 (2002).
Despite the several advantages over the strept/avidin-biotin and bispecific antibody-hapten systems, a few limitations exist in the use of these oligomers in pretargeting. These limitations include poor specificity, possible insolubility in aqueous solutions, and high costs.
A need continues to exist for an improved kit and method for in vivo targeting to deliver a therapeutic or diagnostic agent to a target site in a mammal, that is more specific, affordable and inexpensive and provides higher target uptake and lower uptake in normal tissues.